Sanitized animal bedding material and process

ABSTRACT

The present invention provides compositions and methods for sanitization or sterilization of, or reduction or elimination of microbes from, PFR material, in particular material for use as an animal bedding. In another aspect, the invention provides sanitized or sterilized material, and especially animal bedding material, prepared using the described compositions and methods.

FIELD OF THE INVENTION

The present invention relates generally to the fields of microbialphysiology, specifically relating to microbiology, and to materials forbedding used in the environment of contained animals.

BACKGROUND OF THE INVENTION

Corncob particles that are not sterilized or sanitized (hereinafterreferred to as traditional corncob) and used as animal bedding worldwidehave relatively high microbial populations. High levels of microbialcontamination increase the likelihood that disease-causingmicroorganisms (pathogens) may be present. Pathogens in beddingmaterials can be harmful to animals by infection of wounds or by causingdigestive or respiratory problems and thereby confounding results ofexperiments. Consequently, many animal research facilities controlagainst pathogens in their animal bedding by either purchasing beddingthat has been irradiated or autoclaving bedding that has not beenirradiated.

In the field of animal management, specifically that of laboratoryanimals, such as rodents, all environmental conditions to which theanimals are exposed must be tightly controlled to prevent contaminationsof the animals by the external environment and/or nosocomialcontamination.

Research animals are becoming more valuable because many disease modelsare expensive and time-consuming to develop. Some longitudinal studiesrequire data collection on the same animals over their lifetimes.Preventing nosocomial infection is paramount in maintaining theintegrity of the research design and in preserving valuable laboratorystock for continued study.

Most research institutions invest substantial resources to keep thesevaluable animal assets safe. Microbial safety and cost factors are majorissues associated with use of bedding materials for laboratory animals.Traditional corncob after it enters the lab animal facility leaves openthe possibility that pathogenic bacteria are introduced to the facilityin storage and handling before sterilization efforts. Using irradiatedor pre-sanitized corncob essentially eliminates that risk because thecorncob arrives at the facility with near sterile characteristics.However, the cost of irradiated corncob bedding can triple that ofnon-sanitized bedding, and autoclaving is widely understood to becostly, especially when energy costs are taken into account. The highcosts of irradiating or autoclaving present an opportunity for appliedscience to achieve the same degree or better of near-sterility at asignificantly lower cost.

The destruction of pathogenic bacteria, fungi and viruses in corncobparticles can be achieved by rigorous chemical or physical methodsrequired to destroy bacterial endospores. This is true because bacterialendospores exhibit the highest resistance to chemicals, heat orirradiation compared to other microorganisms including viruses.

Another, less common, method for sterilizing food is the tyndallizationprocess, named after the 19th century scientist John Tyndall.Tyndallization essentially consists of heating the substance for 15minutes for three days in a row (usually by boiling it). During thewaiting periods over the three days, the substance being sterilized iskept at a warm room temperature; i.e., a temperature that is conduciveto germination of the spores. On the second day most of the spores thatsurvived the first day will have germinated into bacterial cells. Thesecells will be killed by the second day's heating. The third day killsbacterial cells from late-germinating spores. This process requiresconsiderable time, and the material being treated must be maintained atthe proper conditions over the entire 3-day period. Further, thetyndallization process is not considered reliably effective.

It is challenging to destroy bacterial endospores with interventionsother than those previously mentioned which are costly, cumbersome,difficult to scale up, and raise questions about reliability. Oneapproach to kill the endospores with greater practicability is to renderthem more susceptible to the inactivation method. One way to decreasespore resistance is to induce spore germination.

Accordingly, the overall goal of the present invention is to provide anovel process to substantially reduce or eliminate populations ofbacterial endospores in corncob particles by exploiting their vulnerablestate—after germination. Once germinated, spores have decreasedresistance to chemicals, heat or irradiation.

Therefore, it is a primary object, feature, or advantage of the presentinvention to improve upon the state of the art.

It is a further objective, feature or advantage of the present inventionto provide methods for substantially reducing or eliminating populationsof bacterial endospores in fibrous material.

It is a further objective, feature or advantage of the present inventionto provide methods for substantially reducing or eliminating populationsof bacterial endospores in plant-fiber-rich (PFR) material by exploitingthe vulnerable state of those spores when stimulated to germinate.

It is a further objective, feature or advantage of the present inventionto provide reliable methods for reducing or eliminating populations ofbacterial endospores in PFR material, wherein the process can becompleted in less than one day, and preferably less than five hours.

It is a further objective, feature or advantage of the present inventionto provide reliable methods for reducing or eliminating populations ofbacterial endospores in PFR material, wherein the product of the processis a dry PFR material.

It is a further objective, feature or advantage of the present inventionto provide PFR material, for example corncob particles, that has beensanitized by a process that substantially reduces or eliminatespopulations of bacterial endospores in corncob particles by exploitingthe vulnerable state of those spores when stimulated to germinate.

It is a further objective, feature or advantage of the present inventionto provide reliable methods for reducing or eliminating viruses in PFRmaterial, wherein the product of the process is a dry fiber-richmaterial.

SUMMARY OF THE INVENTION

The present invention provides methods for sterilizing or substantiallyreducing or eliminating populations of bacterial endospores—the mostresistant microbial life forms—in a material. In one aspect, theinvention involves a method of sterilizing, substantially reducing, oreliminating populations of microbes in PFR material, comprising exposingthe PFR material to a heat-shock of 65° to 90° C. for 10 to 30 minutes;and drying the PFR material by heating it. In one aspect, the PFRmaterial is a bedding material, preferably corncob particles. In apreferred embodiment, the heat shock is about 80° C. for about 15minutes. In another aspect, the heating comprises exposing theheat-shocked PFR material to a temperature of between about 115° and155° C. for between about 25 and about 40 minutes.

In one embodiment, the method also involves adding a germinate to thePFR material prior to heat-shocking, wherein said germinant is effectiveto promote germination of bacterial endospores. In a preferredembodiment, the germinant is corn powder.

In another embodiment, the method also involves wetting the PFR materialwith water prior to heat shocking. In a preferred embodiment, theparticles are coated with water that contains the germinate. In anotherembodiment, the method further involves holding the PFR materialfollowing the heat shocking, wherein said holding comprises incubatingthe PFR material at between about 35° and about 55° C. for 10 to 30minutes. More preferably, the holding step involves maintaining the PFRmaterial at an internal temperature of about 40° C. for at least 20minutes. In a more preferred embodiment, the entire process may beperformed in less than one day, and most preferably in less than fivehours.

In another aspect, the present invention provides PFR material that issubstantially free of microbes produced by the described methods. In apreferred embodiment, the PFR material is a bedding material, morepreferably corncob particles. In a more preferred embodiment, the PFRmaterial produced by the described methods has a viable microbe contentof 10 or less log₁₀ CFU/g of PFR material. In an even more preferredembodiment, the PFR material produced by the described methods has aviable microbe content of 1 or less log₁₀ CFU/g of PFR material. Inanother aspect, the PFR material produced by the described methods isdry.

While multiple embodiments are disclosed, still other embodiments of thepresent invention will become apparent to those skilled in the art fromthe following detailed description, which shows and describesillustrative embodiments of the invention. Accordingly, the drawings anddetailed description are to be regarded as illustrative in nature andnot restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A-B) shows bacterial colony counts from spores that were heatshocked at 80° C. for various times in (A) water or in (B) wet corncobparticles.

FIG. 2 shows aerobic plate count (total count; APC), Enterobacteriaceae(ENT) and yeast and mold (Y&M) counts in wet ⅛″ corncob particles (seeTable 1) before and after heat shock (HS) at 80° C. for 15 minutes.

FIG. 3 shows the effect of holding time at the best holding temperature(40° C.) on germination of bacterial spores in wet artificiallyinoculated ⅛″ corncob particles.

FIG. 4 shows viability of bacterial spores after drying of heat-shocked(HS) and non-heat shocked (NHS) wet corncob particles at 176.6° C. for20 minutes.

FIG. 5 (A-C) shows viable counts of aerobic mesophilic bacteria (APC),Enterobacteriaceae (ENT) and yeast and molds (YM) in naturallycontaminated corncob particles. (A) Viable counts of aerobic mesophilicbacteria, Enterobacteriaceae and yeast and molds in naturallycontaminated corncob particles before heat-shock. (B) Numbers of viableaerobic mesophilic bacteria, Enterobacteriaceae and yeast and molds innaturally contaminated corncob particles after application of heat-shocktreatment of 80° C. for 15 minutes. (C) Numbers of viable aerobicmesophilic sporeformers (AMS) and aerobic thermophilic sporeformers(ATS) in naturally contaminated corncob particles after application ofheat-shock treatment 80° C. for 15 minutes.

FIG. 6 (A-B) shows log₁₀ CFU/g reductions in numbers of viable sporesthat were not heat shocked (NHS) or treated with HST1× or HST2× incorncob particles and heated at 150° C. for 25 to 40 minutes (A) or 155°C. for 25 to 35 minutes (B).

FIG. 7 (A-B) shows bacterial colonies from spores after thermaltreatment (155° C., 25 min) of moist corncob particles (⅛″; 80% (w/w)added water) after heat shock (80° C., 15 min) and tempering at 40° C.for 45 minutes. (A) One heat shock/tempering process (HST1×) beforethermal treatment; (B) Two sequential shock/tempering processes (HST2×)before thermal treatment.

FIG. 8 shows colonies of bacterial sporeformers on dextrose tryptoneagar following incubation at 35° C. for 72 hours.

FIG. 9 (A-D) shows bacterial growth from non-heat shock (NHS; top platein A-C, Control (no heating) in D) and heat shocked corncob particles(HS; bottom two plates in A-C, water, 0.25% corn steep water and 0.1%peptone in D). Corncob particles were coated with water containing theindicated germinate prior to heat shock, and all corncob particles weredried by heating at 155° C. for 25 minutes.

Various embodiments of the present invention will be described in detailwith reference to the drawings, wherein like reference numeralsrepresent like parts throughout the several views. Reference to variousembodiments does not limit the scope of the invention. Figuresrepresented herein are not limitations to the various embodimentsaccording to the invention and are presented for exemplary illustrationof the invention.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Units, prefixes, and symbols may be denoted in their SI accepted form.Numeric ranges recited within the specification are inclusive of thenumbers defining the range and include each integer within the definedrange. The terms defined below are more fully defined by reference tothe specification as a whole.

As used herein, an “animal” is defined to include any organism kept as apet or commonly housed in an animal care or animal housing or vivariumfacilities, and especially animals that are kept in cages, includinglaboratory and research animals. Such animals include rodents, such asmice, rats, hamsters, gerbils, Guinea pigs, ferrets, and rabbits; birdssuch as a quails, chickens, turkeys, parrots, parakeets, canarys, andfinchs; canines, such as domesticated dogs; felines, such asdomesticated cats; and primates such as a monkeys, chimpanzees, rhesusmacaques and gorillas.

“PFR material” refers to any substance that is primarily comprised ofcellulose, hemicelluloses, pectins and/or other water-soluble and-insoluble plant fiber material. PFR material may be any material thatis a component or derivative of an agricultural plant, including, forexample, pressed wood pellets, wood shavings, kenaf, sawdust, wheatstraw, barley straw, oat straw, timothy straw and various forage straws,and corncob particles. Included in, but not limited to, are thefiber-containing materials such as plant tubers, wheat, seed, shells ofseeds, stems, roots, and leaves of plants, fruits and their skins, wood,tree branches, tree bark, straw, grass, and waste materials originatingfrom the agricultural industry, for example, distiller's dry grain,sugar beet pulp, cellulose pulp, paper waste, cotton, linen, vegetablesand vegetable waste, such as tomato skins and seeds, and the like. PFRmaterial may also include cardboard products, such as for examplerecycled cardboard. “Bedding material” as used herein includes any typeof PFR material that is commonly used within the field of animalhusbandry. In a preferred embodiment, the bedding material is composedof corncob particles.

“Corncob particles” refers to a variety of corncob particle componentsof different types and sizes that have been prepared and fractionatedfrom corncobs. Corncob particles may be derived from the cob's densewoody interior ring portion, e.g., in the form of broken granules knownas corncob grit or grit granules or grit particles (or simply “grit”).Corncob particles may also be derived from pith, chaff and beewingportions of the cob. Corncob particles can have various grades, definedby the approximate percent of particles retained on test screens (U.S.Standard), with examples shown in Table 1. Reference to the size ofcorncob particles is based on these designations.

TABLE 1 Approximate percent of particles retained on test screens (U.S.Standard). Screen # 1/4″ 1/8″ 1014 1020 1420 2040 4060 −40 1440 PC −40PC 6 45% 8 50% 10  5% 30% 14 70% 55% 35% 20 45% 60% 75% 30% 30  5% 20%35% 40% 40  5% 60% 10% 30% 10% 50  5% 60 90% 80 10% Pan 90% 90%It is understood that the methods of sterilizing, or reducing oreliminating microbes are effective for all particle sizes. Corncobs arerich in plant fiber and are particularly suitable for bedding material.While other PFR materials might be used, corncobs are particularly wellsuited for the present application because of their absorbency, which isprimarily due to the physico-chemical characteristics of the particles.

“Microbes” as used herein includes, but is not limited to bacteria,fungi, archaea, viruses and protozoans, such as, for example, yeast,molds and bacteria, including sporulating bacteria. Microbes includemicroorganisms naturally present in harvest, processing, storage, andtransport of PFR material, including corncobs and corncob particles.

“Germinant” refers to a nutrient composition material that promotesmicrobial growth. Germinants include those derived fromagricultural-based material such as, for example, baggasse powder,rice-straw powder, wheat bran, corncob powder, and corn powder.Germinants may also include organic nutrients in natural complexes or inisolation, such as, for example, malt extract, yeast extract, potatoextract, inosine, glucose, glycine, L-alanine, L-serine, L-leucine,L-isoleucine, peptone, soya-peptone, bactopeptone, and corn steep water.Corn steep water refers to water to which powdered or pulverized cornhas been introduced, for example, introducing 5 pounds of pulverizedcorn kernels to 2000 pounds of water. Germinants may also includeculture media, such as, for example, lysogeny broth (LB; a.k.a. Luriabroth, Lennox broth, or Luria-Bertani) medium, potato dextrose agar,Sabouraud agar, chocolate agar, nutrient agar, plate count agar, and thelike.

“Substantial” or “substantially” with reference to decreasing, reducingor eliminating a material, including, for example, water or microbesrefers to compositions completely lacking microbes or having such asmall amount of the component that the component does not affect theperformance of the composition. By way of example only, substantiallyeliminating microbes from a material could involve a reduction ofmicrobes to or below the limit of detection of standard measurements.

Methods for Sterilizing, or Substantially Reducing or EliminatingPopulations of Bacterial Endospores in PFR Material

According to one aspect of the invention, methods are provided thatdecrease or eliminate microbes from PFR material, including beddingmaterial. In a preferred embodiment, the processes substantiallyeliminate microbes from the PFR material.

In one embodiment, the method comprises exposing the PFR material to afirst heat shocking step followed by a second heating step. The heatshocking step involves exposing the PFR material to a temperaturebetween about 65° and about 90° C. for 10 to 30 minutes. In a preferredembodiment, the heat shocking comprises exposure at about 80° C. forabout 15 minutes.

According to one embodiment of the invention, the second heating stepinvolves exposing the PFR material at greater than 115° C. In apreferred embodiment, the heating is at greater than 121° C. In a morepreferred embodiment, the heating is between about 150° and 180° C. Theheating step is carried out for a sufficient amount of time toadequately dry the material, depending on the temperature used. Forexample, the heating may be at 115° C. for 40 minutes, or at 155° C. for25 minutes.

In one aspect, the heat shocking step and/or heating step may beperformed using any technique that achieves the necessary temperature.For example, the heating may be performed using a fluidized sand bath, awater bath, a heating element, a conduction heater, an oven, orradiation heating such as infrared, ultraviolet, microwave, radiofrequency, and high-frequency (HF) waves. In a preferred embodiment, theheat shocking step is performed using a water bath. In another preferredembodiment, the second heating step is performed using a forced-airconvection oven/dryer.

In one embodiment, the PFR material may be treated prior to beingsubjected to the heat shocking and heating steps. For example, the PFRmaterial may be wetted or soaked, preferably with water. In a preferredembodiment, the PFR material may also be mixed with a germinant, forexample by coating the PFR material with water containing a germinant.In a more preferred embodiment, the germinant is peptone solution,L-alanine solution, or corn steep water.

In one aspect, the methods may also include a holding step between theheat shocking and second heating steps, wherein the soaked PFR materialis incubated at between about 35° and about 55° C. for 10 to 30 minutes.In a preferred embodiment, the PFR material maintains an internaltemperature of about 40° C. for at least 20 minutes.

Sterilized PFR Material

In another aspect, the invention encompasses PFR material that resultsfrom the described processes, wherein the resulting PFR material issanitized or sterile, and at least substantially free of microbes. In apreferred embodiment, the PFR material is corncob particles. In anotherpreferred embodiment, the viable microbe content of the PFR materialfollowing processing is 10 or less CFU/g of PFR material.

In one aspect, the PFR material is dry flowing treatment by thedescribed process. In a preferred embodiment, the PFR material has lessthan 20% moisture content following the described process. In a morepreferred embodiment, the PFR material has less than 10% moisturecontent following the described process.

The following examples are intended for illustration purposes only andare not intended to limit the invention in any way.

EXAMPLES Example 1 Extent of Germination of Bacterial Spores FollowingHeat-Shock Treatments in Sterile Water and in Sterile Wet CorncobParticles

Bacterial spores are extremely heat resistant. Reliable heatinactivation of bacterial spores can be achieved by autoclaving whichtypically refers to the application of pressurized steam at 121° C., 15psi, for about 20 minutes depending on the quantity of material to betreated. However, autoclaving can be costly, time consuming, and limitedby the volumetric constraints of the autoclave. It is documented thatbacterial spores (if triggered to germinate) exhibit decreasedresistance to heat. Some bacterial spores will not germinate unlessactivated by heat-shock, which breaks spore dormancy. Sub-lethal heating(heat-shock) increases the number of bacterial spores that germinatewithin a spore population. Thus, it is important to determine the extentof germination of bacterial spores following heat-shock treatments insterile water and in sterile wet corncob particles.

Sterilized water and corncob particles (⅛″; see Table 1) were inoculatedwith bacterial spores to give ˜1×10⁵ (5.0 log) spores per ml (water) orper gram (corncob particles). Bacterial spores were harvested fromspore-forming bacteria isolated from traditional corncob particles andused to inoculate water to obtain ˜10⁵ colony forming units (CFU) perml. The water was sterilized by autoclaving and cooled to ambienttemperature (23° C.) before inoculation. Large tubes of water containingbacterial spores were heat-shocked at 65, 70, 80 and 90° C. in athermostatically controlled water bath. The tubes of spore suspensionwere held at each heat-shock temperature for 10, 20, and 30 minutesbefore immersing them in an ice/water mixture. The exposure timerepresents the length of time that the samples have been exposed to theappropriate temperature. The come-up time (time required for samples toreach the appropriate temperature) within the test tube was recorded.Tubes of non-heat-shocked spore suspensions served as control.

Treatment conditions for water (heat-shock temperature and time) werealso used in heat-shock experiments involving corncob particles. Samples(10-gram) of sterile corncob particles were placed in large test tubes.The particles were inoculated with bacterial spores to obtain ˜10⁵CFU/g. In each tube the inoculated particles were soaked with 10 ml ofsterile water and heat-shocked as previously described for bacterialspores in water.

Enumeration of bacterial colonies was performed to estimate the numberof spores that germinated under each heat-shock condition (temperatureand time). Microbial analyses were performed according to standardmethods of analysis adapted from the Compendium of Methods for theMicrobiological Examination of Foods, 4th edition (APHA, 2001). Numbersof spore-forming bacteria from heat-shocked and control spores in wateror corncob particles were counted. Ten-fold serial dilutions of sporesin water or corncob particles were prepared in buffered peptone waterand aliquots of diluted suspension were surface-plated on dextrosetryptone agar (DTA). Inoculated DTA plates were incubated at 35° C. for48 hours (for mesophilic aerobic spore-formers) and 55° C. for 48 to 72hours (for thermophilic aerobic spore-formers).

The extent of germination of bacterial spores following heat-shock ofspores in water and wet corncob particles is shown in FIGS. 1A and 1B.Heat-shock at 80° C. for 10 or 15 minutes resulted in highest bacterialcolony counts from the spores in water or corncob particles. There wereno significant differences in colony counts at heat-shock treatments at80° C. for 10, 15, and 20 minutes.

Heat-shock of spores in water or wet corncob particles at 80° C. for 10to 20 minutes increases the extent of spore germination. This is basedon the observed increase in bacterial colony counts from heat-shockedspores compared to control (23° C.; non-heat-shocked). The fact thatheat-shock at 80° C. for 10 to 20 minutes produced bacterial colonycounts ranging from 4.67 to 4.75 log CFU/ml or g (less than the 5.0 loginitial spore count/ml or g) indicates that not all the sporesgerminated.

The length of time between heat-shock and germination of bacterialspores can vary among types of spores. Also, the optimal temperature forthe germination process following heat-shock can vary among spores.Therefore, it is important to determine the influence of various postheat-shock holding times and temperature on the extent of germination ofbacterial spores in corncob particles to further increase the extent ofspore germination.

Example 2 Enterobacteriaceae, Yeast, Molds and Spore-Forming BacteriaViability in Heat-Shocked and Non-Heat-Shocked Corncob Particles

Corncob particles, like many raw agricultural products, are contaminatedwith organisms (other than spore-forming bacteria) such asEnterobacteriaceae, yeast and molds. One or more temperature/timecombinations used for heat-shock may kill these microorganisms to reducethe microbial load of corncob particles.

To determine whether vegetative bacterial cells, yeast, and molds arecapable of surviving the heating conditions used for heat-shock ofbacterial spores in corncob particles, heat-shock temperature and timethat produce the largest amount of spores that germinate (based on platecounts of spore-formers) were used in experiments to determine theeffect of this procedure on the natural microbial content of traditionalcorncob particles. Enumeration of microbial groups (bacteria and fungi)was performed before and after heat-shock treatment. Briefly, theparticles from each tube were aseptically transferred to separatesterile 250-ml screw-cap Erlenmeyer flasks. To each flask, 80 ml ofsterile 0.1% (w/v) peptone were added. The flasks were vigorously shakento remove microbial cells from the particles. Aliquots (0.1-ml or1.0-ml) of wash solution were plated on appropriate agar media todetermine numbers of viable microorganisms. Samples (10-gram) of sterilecorncob particles were placed in large test tubes. In each tube theparticles were soaked with 10 ml of sterile water and heat-shocked aspreviously described for bacterial spores in water.

Microbial analysis of corncob particles (heat-shocked andnon-heat-shocked) was performed as previously described. Appropriatenutrient agar plates, plating technique and incubation conditions usedto obtain viable counts of specific microbial groups are provided inTable 2.

TABLE 2 Agar media, plating technique, and incubation conditions formicrobiological tests to be performed on heat-shocked andnon-heat-shocked corncob particles Plating Incubation Microbial TestAgar Media Technique Conditions Aerobic plate count PCA Surface plate30° C. (86° F.), 48 hours Enterobacteriaceae TSA/VRB Pour plate 35° C.(95° F.), 24 overlay hours Mesophilic aerobic DTA Surface plate 35° C.(95° F.), 24 spore-formers hours Thermophilic aerobic DTA Surface plate55° C. (131° F.), 48- spore-formers 72 hours Yeast and molds DRBC agarSurface plate 25° C. (77° F.), 5 days PCA = plate count agar; DTA =dextrose tryptone agar; TSA = tryptic soy agar; VRB = violet red bileagar; DRBC = dichloran rose bengal chloramphenicol agar

Aerobic plate count (total count) and counts of Enterobacteriaceae (ENT)and fungi (yeast and molds—YM) following heat-shock (80° C. for 15minutes) in wet corncob particles are shown in FIG. 2. Heat-shocktreatment of the wet particles decreased the aerobic plate count byapproximately 0.97 log CFU/g. Numbers of viable ENT were below thedetection limit (<10 CFU/g) in both heat-shocked and control samples.Viable YM were destroyed by the heat-shock treatment; none were detectedin heat-shocked particles.

The aerobic plate count of the corncob particles gives an estimate ofall viable vegetative cells that are able to grow aerobically under theconditions (agar medium, and incubation temperature and time) used inthe present study. Bacterial spores that germinate (without heat-shock)and produce vegetative cells that form colonies on the agar medium alsocontribute to the aerobic plate count of non-heat-shocked particles.

Vegetative cells of bacteria and fungi are easily killed by temperaturesthat are merely used to heat-shock bacterial spores. Therefore, thedecrease in aerobic plate count (0.97 log CFU/g) in the corncobparticles after heat-shock at 80° C. for 15 minutes is likely due todeath of the more susceptible vegetative cells. All the bacterialcolonies on agar-plated heat-shocked samples were from spore-formingbacteria. This is not surprising because bacterial spores make up asubstantial part of the microbial population of dried corncob particles.

The absence or very low numbers (<10 CFU/g) of Enterobacteriaceae in thecontrol corncob particles indicate that intestinal pathogens such asSalmonella enterica, Shigella, and Escherichia coli are absent.Enterobacteriaceae was isolated from some of the whole corncobs, but notfrom the ground corncob particles (⅛″) in the batch provided for thepresent study.

Contamination of whole corncobs with Enterobacteriaceae from feces ofbirds, rodents or insects that frequent the corncob piles might besporadic, and contaminating microbes are likely diluted out during theprocessing of the corncobs to produce the particles. Also, some of thoseorganisms probably died from the very harsh dry conditions ofprocessing.

Like bacterial vegetative cells, many yeast and molds are easilydestroyed by temperatures used to heat-shock bacterial spores. Resultsof the present study suggest that contaminating fungi in corncobparticles could be eliminated during the heat-shock part of themanufacturing process for sanitized corncob particles.

Example 3 Effect of Holding Temperature and Time (Post Heat-Shock) onthe Extent of Germination of Bacterial Spores in Corncob Particles

After bacterial spores are heat-shocked, the length of time for thestart of germination can vary among spores in a population. Also, theideal post-heat-shock temperature for germination may vary depending onwhether the spores are mesophilic or thermophilic. Since both groups ofspores are present in corncob particles, it was important to determinetemperature/time conditions that produced the maximum amount ofgerminating spores of both groups to enhance their destruction.

The extent of germination of heat-shocked bacterial spores is dependenton post heat-shock conditions of temperature and length of time at aspecified temperature. Sterile water and corncob particles wereinoculated with bacterial spores and heat-shocked as described above.Following heat-shock, the samples were placed in a 50/50 ice/watermixture. Temperature of the samples of water and corncob particles wasmonitored using thermocouples placed in separate tubes of non-inoculatedsamples. When internal temperature of the samples reached 35°, 40°, 45°,or 55° C., the tubes were transferred to water baths (set at appropriatetemperatures) and held at a specific temperature for 0 (control), 10, 20or 30 minutes before performing microbial analysis.

Ten-fold serial dilutions of spores in water or corncob particles wereprepared in buffered peptone water and aliquots of diluted suspensionwere surface-plated on dextrose tryptone agar (DTA). Inoculated DTAplates were incubated at 35° C. for 48 hours (for mesophilic aerobicspore-formers) and 55° C. for 48 to 72 hours (for thermophilic aerobicspore-formers).

Results are shown in FIG. 3. The post heat-shock conditions of 40° C.(for 20 minutes or longer) resulted in the highest amount of sporegermination compared to other temperature and time conditions used inthe present study. At a holding temperature of 40° C., there were nosignificant differences in the extent of spore germination at 20, 30 or35 minutes.

When bacterial spores are heat-shocked to trigger germination, theirenzymes become active and set the process of spore germination inaction. Since enzyme activity is affected by temperature, certaintemperatures may stop, slow down or increase the spore germinationprocess. Therefore, to optimize germination, the best post-heat-shockholding temperature and time needed to be determined. Based on theseresults, cooling the heat shocked corncob particles to 40° C. andholding them at that temperature for a minimum time of 20 minutes werethe best conditions for increasing the extent of germination ofbacterial spores in wet ⅛″ corncob particles.

Example 4 Viability of Bacterial Spores after High Temperature Drying ofHeat-Shocked and Non-Heat Shocked Corncob Particles

To determine whether heat-shocked bacterial spores in corncob particlesare more readily destroyed by high temperature drying compared tonon-heat-shocked spores, optimized conditions of heat-shock andpost-heat-shock holding temperature and time obtained from experimentsdescribed in Examples 1, 2, and 3, were used to determine the viabilityof bacterial spores after high temperature drying of heat-shocked andnon-heat-shocked (control) corncob particles.

Corncob particles (⅛″) were sterilized by autoclaving. The sterilizedparticles were inoculated with bacterial spores to give ˜1×10⁵ (5.0 log)spores per gram. Spores in corncob particles were heat-shocked at 80° C.for 15 minutes as previously described. Following heat-shock theinoculated particles were cooled to 40° C. and held at that temperaturefor 20 minutes before drying them at 176.6° C. for about 20 minutes.

Fifty-gram portions of wet corncob particles that had been subjected toheat-shock or non-heat-shocked control were placed in separate dishes.Both sets of particles were dried by heating in a forced-air convectionoven/dryer at 176.6° C. for approximately 15 to 20 minutes. Theparticles were held for 30 minutes at ambient temperature in a laminarflow hood before analyzing them for numbers of viable bacterial sporesand microbial vegetative cells. Particles that were not dried in theoven served as control and were used to determine the initial sporecount in the corncob particles. The control and oven-dried particleswere held for 30 minutes at ambient temperature (23° C.±1° C.) in alaminar flow hood before analyzing them for numbers of viable bacterialspores based on bacterial colony counts on dextrose tryptone agar.

Twenty five-gram samples of corncob particles were aseptically placedinto separate sterile stomacher bags and mixed with buffered peptonewater (225 ml per sample). The mixtures were vigorously shaken by handfor 30 seconds and ten-fold serial dilutions of the wash solution wereprepared in 0.1% peptone water. Aliquots (1.0- or 0.1-ml) from the washsolutions or from selected dilutions of the wash solutions were platedonto appropriate agar media to determine the numbers of microorganisms(including spore-formers) in the particles.

In corncob particles that were not oven-dried, numbers of bacterialcolonies from heat-shocked spores were higher than those fromnon-heat-shocked spores. Numbers of bacterial colonies fromnon-heat-shocked spores in corncob particles remained relativelyconstant during the drying process. Viable counts were 3.82, 3.78, and3.80 log CFU/g, in control particles (not oven-dried), oven-driedparticles (Rep 1) and oven-dried particles (Rep 2), respectively (FIG.4).

The viability of heat-shocked (80° C., 15 minutes) bacterial sporeswhich were held at 40° C. for 20 minutes, was destroyed duringoven-drying at 176.6° C. for ˜20 minutes. Over two trials of theexperiment viable bacterial counts were beyond the detection limit (<10CFU/g).

Based on the higher numbers of bacterial colonies that resulted fromheat-shocked spores compared to those of non-heat-shocked spores, theheat-shocking of bacterial spores in corncob particles under conditionsused herein increased the extent of spore germination.

Complete destruction of non-germinating bacterial spores cannot beachieved by the drying temperature and time (176.6° C. for ˜20 minutes)described here. This is supported by data presented herein showing thatthe spore-forming bacterial count in corncob particles (withnon-heat-shocked spores) was not changed during drying (FIG. 4). Incontrast, viable bacterial counts were below the level of detection inparticles that contained heat-shocked spores and subjected to those samedrying conditions. These results indicate that these heat-shockconditions and post-heat-shock holding conditions can sensitizebacterial spores to heat and thus inactivate the spores during drying ofcorncob particles at 176.6° C. for ˜20 minutes.

Example 5 Optimizing Germination and Heat Destruction of BacterialSpores to Sanitize PFR Material

The results of the above examples demonstrate the efficacy of thedescribed sanitization method. However, the Inventors sought to optimizethe process. Accordingly, the present example describes the optimizationof heat-shock parameters on the natural microbial content of traditionalcorncob particles, and the minimum oven drying temperature required toachieve destruction of heat-shocked bacterial spores in naturallycontaminated corncob particles.

Traditional corn cob particles were subjected to heating at 80° C. for15 minutes to heat-shock bacterial spores. Before heat treatment, thecorncob particles were wetted with filter-sterilized water to obtaininitial amounts of added water ranging from 30% to 65% (w/w). Afterheat-shock, the particles were tempered at 40° C. (104° F.) for ˜20 to25 minutes before drying them in a convection oven/dryer.

To evaluate the effect of oven drying temperature, fifty-gram portionsof wet corncob particles (heat-shocked and non-heat-shocked control)were placed in separate sterile aluminum trays. Both sets of particleswere dried by heating in a forced-air convection oven/dryer at 82, 93,104, 115, 121 and 155° C. During heating at a specified temperature theparticles were removed at set times (20, 25, 30, 35, and 40 minutes),tempered to ˜22±1° C. for 30 minutes in a laminar flow biological hood(with the fan on), then analyzed for numbers of viable bacterial sporesbased on colony counts of spore-forming bacteria.

To evaluate the effect of heat-shock treatment on the numbers of viablebacteria in corncob particles, samples of particles were analyzed forthe following microbial groups: aerobic mesophilic bacteria,Enterobacteriaceae, yeast and molds, aerobic mesophilic spore-formersand aerobic thermophilic spore-formers before and after heat-shock, asdescribed above.

FIG. 5A shows numbers of viable aerobic mesophilic bacteria,Enterobacteriaceae, and yeast and molds in naturally contaminatedcorncob particles (⅛″) before application of heat-shock treatment.Viable numbers of aerobic mesophilic bacteria, Enterobacteriaceae andyeast and mold, ranged from 4.71 to 4.84, 3.42 to 3.98, and 3.30 to 3.63log₁₀ CFU/g of corncob particles, respectively.

FIG. 5B shows numbers of viable aerobic mesophilic bacteria,Enterobacteriaceae, and yeast and molds in naturally contaminatedcorncob particles (⅛″) after application of heat-shock treatment (80° C.for 15 minutes). Viable numbers of aerobic mesophilic bacteria rangedfrom 4.02 to 4.29 log₁₀ CFU/g of corncob particles. NoEnterobacteriaceae or yeast and molds were detected following heatshock.

FIG. 5C shows numbers of viable aerobic mesophilic spore-formers andaerobic thermophilic spore-formers in naturally contaminated corncobparticles (⅛″) after application of heat-shock treatment (80° C. for 15minutes). Levels of thermophilic spore-formers in naturally contaminatedcorncob particles (⅛″) were consistently lower than those of mesophilicspore-formers. Numbers of viable aerobic mesophilic spore-formers andaerobic thermophilic spore-formers, in heat-shocked particles rangedfrom 4.07 to 4.33 and 3.69 to 3.90 log₁₀ CFU/g, respectively.

Tables 3 to 10 show the effects of drying temperature and time on sporeviability of aerobic mesophilic bacteria in non-heat-shocked (NHS) andheat-shocked (HS) naturally contaminated corncob particles (⅛) with 30to 65% (w/w) added water. Non-heat-shocked spores (mesophilic) weretotally unaffected by the heat applied for drying of the corncobparticles. Similar results were obtained for thermophilic spores (datanot shown)

For ease of reference, areas highlighted in grey indicate temperatureand time parameters (at a specified level of water initially added tothe particles) at which no bacterial survivors were detected based onthe plate count method. The culture method used had a detection limit of10 CFU/g of corncob particles.

TABLE 3 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 30% (w/w) added water. Temper- Prior ature heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min 82° C. NHS +++ +++ +++ ++ + +++ HS +++ +++ +++ ++ + +++ 93°C. NHS +++ +++ +++ ++ + +++ HS +++ +++ +++ ++ + +++ 104° C. NHS +++ ++++++ ++ + +++ HS +++ +++ +++ ++ + +++ 115° C. NHS +++ +++ +++ ++ + +++ HS+++ +++ +++ ++ + +++ 121° C. NHS +++ +++ +++ ++ + +++ HS +++ +++ +++++ + +++ 176.6° C. NHS +++ +++ n/a n/a n/a HS +++ +++ n/a n/a n/aNon-heat-shocked (NHS) and heat-shocked (HS) Detection (+) or nodetection (−) of viable spores based on bacterial colony counts Eachsign (+ or −) represents results of one replication of the experimentn/a = not applicable; no data collected.

TABLE 4 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 35% (w/w) added water. Temper- Prior ature heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min 82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++ 93° C.NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++ 104° C. NHS +++ +++ ++++++ +++ HS +++ +++ +++ +++ +++ 115° C. NHS +++ +++ +++ +++ +++ HS ++++++ +++ +++ ++− 121° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +−−176.6° C. NHS +++ +++ n/a n/a n/a HS ++− +−− n/a n/a n/aNon-heat-shocked (NHS) and heat-shocked (HS) Detection (+) or nodetection (−) of viable spores based on bacterial colony counts Eachsign (+ or −) represents results of one replication of the experimentn/a = not applicable; no data collected.

TABLE 5 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 40% (w/w) added water. Temperature Prior heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min   82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   93°C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   104° C. NHS +++ ++++++ +++ +++ HS +++ +++ +++ +++ ++−   115° C. NHS +++ +++ +++ +++ +++ HS+++ +++ +++ +−−

  121° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++

176.6° C. NHS

n/a n/a n/a HS ++− +−− n/a n/a n/a Non-heat-shocked (NHS) andheat-shocked (HS) Detection (+) or no detection (−) of viable sporesbased on bacterial colony counts Each sign (+ or −) represents resultsof one replication of the experiment n/a = not applicable; no datacollected.

TABLE 6 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 45% (w/w) added water. Temperature Prior heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min   82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   93°C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   104° C. NHS +++ ++++++ +++ +++ HS +++ +++ +++ ++− +−−   115° C. NHS +++ +++ +++ +++ +++ HS+++ +++ +++

  121° C. NHS +++ +++ +++ +++ +++ HS +++ +++ ++−

176.6° C. NHS +++ +++ n/a n/a n/a HS

n/a n/a n/a Non-heat-shocked (NHS) and heat-shocked (HS) Detection (+)or no detection (−) of viable spores based on bacterial colony countsEach sign (+ or −) represents results of one replication of theexperiment n/a = not applicable; no data collected.

TABLE 7 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 50% (w/w) added water. Temperature Prior heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min   82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   93°C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   104° C. NHS +++ ++++++ +++ +++ HS +++ +++ +++ ++−

  115° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++

  121° C. NHS +++ +++ +++ +++ +++ HS +++ +++ ++−

176.6° C. NHS +++ +++ n/a n/a n/a HS

n/a n/a n/a Non-heat-shocked (NHS) and heat-shocked (HS) Detection (+)or no detection (−) of viable spores based on bacterial colony countsEach sign (+ or −) represents results of one replication of theexperiment n/a = not applicable; no data collected.

TABLE 8 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 55% (w/w) added water. Temperature Prior heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min   82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   93°C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   104° C. NHS +++ ++++++ +++ +++ HS +++ +++ +++ ++−

  115° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++

  121° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +−−

176.6° C. NHS +++ +++ n/a n/a n/a HS

n/a n/a n/a Non-heat-shocked (NHS) and heat-shocked (HS) Detection (+)or no detection (−) of viable spores based on bacterial colony countsEach sign (+ or −) represents results of one replication of theexperiment n/a = not applicable; no data collected.

TABLE 9 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 60% (w/w) added water. Temperature Prior heat- Dryingtime at specified temperature of Drying shock 20 min 25 min 30 min 35min 40 min   82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   93°C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   104° C. NHS +++ ++++++ +++ +++ HS +++ +++ +++ ++−

  115° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++

  121° C. NHS +++ +++ +++ +++ +++ HS +++ +++

176.6° C. NHS +++ +++ n/a n/a n/a HS

n/a n/a n/a Non-heat-shocked (NHS) and heat-shocked (HS) Detection (+)or no detection (−) of viable spores based on bacterial colony countsEach sign (+ or −) represents results of one replication of theexperiment n/a = not applicable; no data collected.

TABLE 10 Influence of drying temperature and time on the spore viabilityof aerobic mesophilic bacteria in naturally contaminated corncobparticles with 65% (w/w) added water Temperature Prior heat- Drying timeat specified temperature of Drying shock 20 min 25 min 30 min 35 min 40min   82° C. NHS +++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   93° C. NHS+++ +++ +++ +++ +++ HS +++ +++ +++ +++ +++   104° C. NHS +++ +++ +++ ++++++ HS +++ +++ +++ +−−

  115° C. NHS +++ +++ +++ +++ +++ HS +++ +++ ++−

  121° C. NHS +++ +++ +++ +++ +++ HS +++ +++

176.6° C. NHS +++ +++ n/a n/a n/a HS

n/a n/a n/a Non-heat-shocked (NHS) and heat-shocked (HS) Detection (+)or no detection (−) of viable spores based on bacterial colony countsEach sign (+ or −) represents results of one replication of theexperiment n/a = not applicable; no data collected.

Example 6 Evaluation of Final Moisture Content of Corncob Particles

In order to determine the effect of various heat-shock, tempering andheating treatment parameters on the moisture content of PFR material,corncob particles (⅛″) were subjected to heating at 80° C. for 15minutes to heat-shock naturally occurring bacterial spores. Prior toheat-shock treatment, the corncob particles were sprayed withfilter-sterilized water to obtain initial amounts of added water of 40,45, 50, 60, 70, 80 or 100% (w/w). After heat-shock, the particles weretempered at 40° C. for 30 minutes in a heater water bath set at 40° C.before drying them in a convection oven/dryer.

Duplicate fifty-gram portions of wet corncob particles that were exposedto heat-shock conditions for bacterial spores and tempered were thenplaced in separate sterile aluminum trays. The particles were heated ina forced-air convection oven/dryer at 115, 121, 150 or 155° C. forselected time periods ranging from 20 to 40 minutes. The heatedparticles were cooled at room temperature for 10 minutes in a laminarflow hood (23±1° C.) with the blower activated. One 50-g batch ofcorncob particles was aseptically divided into two 25-g portions andused for microbial analysis to determine numbers of viable aerobicmesophilic spores in the particles as described above. The remaining50-g samples were used to determine their moisture content.

The corncob samples were weighed and the initial weight of each samplewas recorded. The final moisture content of the particles was determinedby drying them to a constant weight in a convection oven set at 155° C.and monitoring the change in weight at set time intervals (20, 25, 30,and 35 minutes). The particles were considered dried when the weightchange between dryings was less than 0.5 gram. The dried samples werecooled at room temperature (22±1° C.) for 10 to 15 minutes in a laminarflow hood with the blower activated. The samples were weighed again andthe amount of moisture lost was calculated by subtracting the finalweight of a sample from its initial weight. The following equation wasused to calculate the final percent moisture:

${{Final}\mspace{14mu}{percent}\mspace{14mu}(\%)\mspace{14mu}{moisture}} = {\frac{{weight}\mspace{14mu}{of}\mspace{14mu}{moisture}\mspace{14mu}{lost}\mspace{14mu}({grams})}{{Initial}\mspace{14mu}{weight}\mspace{14mu}{of}\mspace{14mu}{corncob}\mspace{14mu}{particles}\mspace{14mu}({grams})} \times 100}$Water activity (a measure of the amount of free or “unbound” moisture)of the corncob particles was measured using an Aqua Lab water activitymeter. As a reference, a water activity value of 1.000 was obtained fordistilled water.

Before they were dried to a constant weight, traditional corncobparticles (⅛″) had a water activity (aW) of 0.364. The moisture contentand water activity of the corncob particles after being dried to aconstant weight averaged 8.33% and 0.273, respectively. Final moisturecontent of the corncob particles decreased with increase in dryingtemperature and time.

Non-heat-shocked (NHS) bacterial spores were not destroyed by any of thetemperature and time combinations used in the present study. Generally,increased amounts added moisture resulted in an increased sensitivity ofthe heat-shocked (HS) spores. For example, with increased amounts ofadded moisture, the heat-shocked spores were destroyed at shorterexposure times at drying temperatures of 115° C., 121° C. and 150° C.

There were no marked differences in the effect of 25 minutes or 30minutes of tempering time on destruction of the spores; however, atempering time of 45 minutes consistently resulted in increaseddestruction of the bacterial spores during drying of the corncobparticles. This effect might be due to the fact that a “wave” ofgermination occurs in a spore population, and not all spores are at thesame point in the germination process at a given time. Increasing thetempering time most likely increases the amount of germinating spores.

TABLE 11 Water activity of non-treated (control) corncob particles(1/8″). Corncob particle Sample weight Water sample no. (grams) Activity(a_(W)) 1 1.00 0.364 2 1.01 0.363 3 1.00 0.363 4 1.03 0.365 5 1.00 0.363Average values 1.01 0.364

TABLE 12 Moisture content and water activity (aw) of non-treated corncobparticles (1/8″) as determined by drying particles to a constant weightin a laboratory scale convection oven set at 155 °C. Weight Weight ofCalculated Corncob Initial Drying after moisture moisture Water particleWeight time drying lost content activity sample no (grams) (min) (grams)(grams) (%) (aW) Rep 1 1 50.13 20 45.98 4.15 8.28 0.284 2 50.05 25 45.904.13 8.26 0.281 3 50.16 30 45.98 4.18 8.34 0.278 4 50.02 35 45.86 4.168.33 0.273 Rep 2 1 50.00 20 45.87 4.13 8.27 0.280 2 50.22 25 46.05 4.178.30 0.278 3 50.10 30 45.95 4.15 8.29 0.271 4 50.18 35 45.99 4.19 8.350.274 Rep 3 1 50.04 20 45.93 4.11 8.22 0.282 2 50.02 25 45.88 4.14 8.280.280 3 50.12 30 45.96 4.16 8.30 0.275 4 50.19 35 45.92 4.17 8.32 0.270Average moisture content = 8.33% Average water activity = 0.272

TABLE 13 Effect of drying temperature and time on the moisture content(%) of corncob particles with 40% (w/w) added water. Temperature Aerobicof Drying mesophilic spores 20 min 25 min 30 min 35 min 40 min 115 ° C.% Moisture Content 10.94 10.61 9.76 9.40 9.05 Viable NHS ++ ++ ++ ++ ++spores HS ++ ++ ++ ++

121 ° C. % Moisture Content 10.86 10.78 9.63 9.28 8.96 Viable NHS ++ ++++ ++ ++ spores HS ++ ++ ++

150 ° C. % Moisture Content 9.34 8.75 8.28 7.92 nd Viable NHS ++ ++ ++++ spores HS ++

155 ° C. % Moisture Content 8.46 8.39 7.89 7.68 nd Viable NHS ++ ++ ++++ spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts nd = no data collected; NHS = no heat shock; HS = heat shock

TABLE 14 Effect of drying temperature and time on the moisture content(%) of corncob particles with 45% (w/w) added water. Temperature Aerobicof Drying mesophilic spores 20 min 25 min 30 min 35 min 40 min 115° C. %Moisture Content 11.63 11.06 10.73 10.24 9.87 Viable NHS ++ ++ ++ ++ ++spores HS ++ ++ ++

121° C. % Moisture Content 11.30 10.84 10.47 9.88 9.65 Viable NHS ++ ++++ ++ ++ spores HS ++ ++ ++

150° C. % Moisture Content 9.26 8.91 8.55 8.30 nd Viable NHS ++ ++ ++ ++spores HS

155° C. % Moisture Content 8.84 8.22 8.00 7.89 nd Viable NHS ++ ++ ++ ++spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts nd = no data collected; NHS = no heat shock; HS = heat shock

TABLE 15 Effect of drying temperature and time on the moisture content(%) of corncob particles with 50% (w/w) added water. Temperature Aerobicof Drying mesophilic spores 20 min 25 min 30 min 35 min 40 min 115° C. %Moisture Content 11.85 11.28 10.94 10.46 10.12 Viable NHS ++ ++ ++ ++ ++spores HS ++ ++ ++

121° C. % Moisture Content 11.59 11.20 10.82 10.21 Viable NHS ++ ++ ++++ ++ spores HS ++ ++ ++

150° C. % Moisture Content 9.48 9.17 9.04 8.60 nd Viable NHS ++ ++ ++ ++spores HS

155° C. % Moisture Content 9.16 8.78 8.53 8.30 nd Viable NHS ++ ++ ++ ++spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts nd = no data collected; NHS = no heat shock; HS = heat shock

TABLE 16 Effect of drying temperature and time on the moisture content(%) of corncob particles with 60% (w/w) added water. Temperature Aerobicof Drying mesophilic spores 20 min 25 min 30 min 35 min 40 min 115 ° C.% Moisture Content 11.96 11.63 11.38 10.96 10.68 Viable NHS ++ ++ ++ ++++ spores HS ++ ++

121 ° C. % Moisture Content 11.86 11.58 11.21 10.85 Viable NHS ++ ++ ++++ ++ spores HS ++ ++

150 ° C. % Moisture Content 10.85 10.72 10.25 9.76 nd Viable NHS ++ ++++ ++ spores HS

155 ° C. % Moisture Content 10.69 10.38 9.87 9.43 nd Viable NHS ++ ++ ++++ spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts nd = no data collected; NHS = no heat shock; HS = heat shock

TABLE 17 Effect of drying temperature and time on the moisture content(%) of corncob particles 70% (w/w) added water. Temperature Aerobic ofDrying mesophilic spores 25 min 30 min 35 min 40 min 115 ° C. % MoistureContent 12.28 12.01 11.73 11.40 Viable NHS ++ ++ ++ ++ spores HS ++

121 ° C. % Moisture Content 12.01 11.68 11.33 11.97 Viable NHS ++ ++ ++++ spores HS ++

150 ° C. % Moisture Content 11.42 11.14 10.82 10.48 Viable NHS ++ ++ ++++ spores HS

155 ° C. % Moisture Content 11.12 10.64 9.98 9.67 Viable NHS ++ ++ ++ ++spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts; NHS = no heat shock; HS = heat shock

TABLE 18 Effect of drying temperature and time on the moisture content(%) of corncob particles with 80% (w/w) added water. Temperature Aerobicmesophilic of Drying spores 25 min 30 min 35 min 40 min 115 ° C. %Moisture Content 12.69 12.36 12.02 11.71 Viable NHS ++ ++ ++ ++ sporesHS ++

121 ° C. % Moisture Content 12.58 12.24 11.90 11.62 Viable NHS ++ ++ ++++ spores HS ++

150 ° C. % Moisture Content 11.84 11.52 11.16 10.66 Viable NHS ++ ++ ++++ spores HS

155 ° C. % Moisture Content 11.26 10.82 10.21 9.88 Viable NHS ++ ++ ++++ spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts; NHS = no heat shock; HS = heat shock

TABLE 19 Effect of drying temperature and time on the moisture content(%) of corncob particles with 100% (w/w) added water. TemperatureAerobic of Drying mesophilic spores 25 min 30 min 35 min 40 min 115 ° C.% Moisture Content 12.82 12.58 12.26 11.93 Viable NHS ++ ++ ++ ++ sporesHS ++

121 ° C. % Moisture Content 12.80 12.47 12.02 11.86 Viable NHS ++ ++ ++++ spores HS ++

150 ° C. % Moisture Content 11.98 11.69 11.27 10.85 Viable NHS ++ ++ ++++ spores HS

155 ° C. % Moisture Content 11.49 10.97 10.46 9.97 Viable NHS ++ ++ ++++ spores HS

Presence (+) or absence (−) of viable spores based on bacterial colonycounts; NHS = no heat shock; HS = heat shock

Example 7 Optimizing the Thermal Destruction of Bacterial Spores in PFRMaterial

Based on results of the above examples, applying this sporicidaltreatment drastically reduced the numbers of viable bacterial spores incorncob particles (⅛″) by ˜10,000-fold (4.0-log). While substantialnumbers of spores can be consistently destroyed by this process, on manyoccasions it was noticed that a small population of spores (about 3.0 to8.0×10¹ spores/g) survived the process even at the highest thermaltreatment applied (155° C. for 25 minutes). This observation was basedon bacterial colonies that emerged on agar plates after 48 hours ofincubation (at ˜60 to 72 hours).

Although in standard methods of microbial analysis (for the aerobicplate count), 48 hours of incubation of inoculated agar plates aretypically used, the emergence of bacterial colonies on agar plates after48 hours, if noticed by the testing laboratory, can markedly impact theresults reported. In this regard, reports on substantial numbers ofbacterial survivors in the treated corncob particles can “weaken” claimsthat the particles are of very high microbial quality and result in lossof acceptance by animal research laboratories.

To address the possibility that some spores were not triggered togerminate by one heat shock, a number of modifications to thesanitization process were assessed in order to optimize destruction ofbacterial spores in PFR material. One approach was to add a second heatshock treatment before final thermal treatment used for drying theparticles to determine if this approach could further reduce the numbersof spore survivors.

A second approach was to evaluate differences in viability of types ofbacterial spores (isolated from corncob particles) and exposed toheat-shock, tempering, and thermal treatment. Since most spores werebeing killed by the process, an additional hypothesis was heatresistance varied among spores present in the corncob particles. In thisregard, we decided to test this latter hypothesis first before exploringthe aspect of germination.

A third approach was to evaluate the effects of water with selectedgerminant solutions, or corn steep water, on the extent of sporegermination in corncob particles. It is known that in addition toadequate moisture plus ideal ranges in heat-shock temperature and time,spores may need certain nutrients (germinants) to initiate germination.This led us to explore the use of several substances in the water usedfor moistening the corncob particles prior to heat-shock.

Influence of Two Heat-Shock and Tempering Cycles on Thermal Destructionof Bacterial Spores in Corncob Particles

Traditional corncob particles (⅛″) with 80% or 100% (w/w) added moisturewere exposed to 80° C. for 15 minutes to heat-shock bacterial spores andkill bacterial vegetative cells. Before heat-shock treatment, thecorncob particles were soaked with filter-sterilized water to obtain 80%or 100% (w/w) added moisture. After each heat-shock, the particles weretempered at 40° C. for 45 minutes. This process was repeated twicebefore drying the corncob particles in a convection oven/dryer. Corncobparticles that were not heat-shocked but soaked with water and heated ina convection oven served as control.

Separate portions (25-g) of wet corncob particles: i) No heat-shock(NHS; control), ii) heat-shock/tempering once (HST1×), and iii)heat-shock/tempering twice (HST2×) were placed in separate sterilealuminum trays. All three sets of particles were dried by heating in aforced-air convection oven/dryer at 150 and 155° C.

During heating at a pre-determined temperature the particles wereremoved at set times (25, 30, 35, and 40 minutes), tempered to ˜22±1° C.for 30 minutes in a laminar flow biological hood (with the fan on), thenanalyzed for numbers of viable bacterial spores based on colony countsof spore-forming bacteria.

Microbial analysis of corncob particles was performed using standardmethods as described above. Inoculated DTA plates were incubated at 35°C. for 72 hours (for mesophilic aerobic spore-formers).

Two replications of each experiment were conducted with two corncobsamples analyzed per treatment per replication. The average counts ofviable microorganisms (transformed to log₁₀ CFU/g) were recorded. Foreach replicate experiment, when bacterial survivors were detected, theaverage log₁₀ CFU/g was used to represent numbers of viable sporeforming bacteria. Because the detection limit was 1.0×10¹ CFU/g, thevalue of <10 was used when no bacterial colonies were detected on agarplates from the lowest dilution (10⁴) of the corncob samples.

The effect of one or two sequential heat-shock/tempering processes onthe heat destruction of bacterial spores in corncob particles (⅛″) with80% (w/w) added water are shown in Table 20.

TABLE 20 Influence of one or two sequential heat-shock/ temperingprocesses on the heat destruction of bacterial spores in corncobparticles with 80% (w/w) added water. Drying Heat-shock and Temperaturetempering 25 min 30 min 35 min 40 min Rep 1 150° C. NHS (control) 4.133.63 3.18 2.79 HST1x 1.78 1.48 1.30 <1.0 HST2x 1.60 1.54 1.00 <1.0 155°C. NHS (control) 4.22 3.75 3.36 nd HST1x 1.74 1.30 1.00 nd HST2x 1.701.48 1.00 nd Rep 2 150° C. NHS (control) 4.24 3.72 3.29 2.71 HST1x 1.901.69 1.48 1.0 HST2x 1.84 1.48 1.30 <1.0 155° C. NHS (control) 4.18 3.983.80 nd HST1x 1.54 1.39 1.00 nd HST2x 1.48 1.39 1.30 nd Counts of viablebacterial spores are expressed as log₁₀ CFU/g. Bacterial colonies werecounted at 72 hours of incubation of agar plates No heat-shock (NHS;control); one heat-shock/ tempering (HST1x); heat-shock/tempering twice(HST 2x ); n/a = not applicable; nd = no data collected

The initial numbers of viable spores in the corncob particles beforeapplication of the sporicidal process were ˜5.20 log₁₀ CFU/g based onresults of microbial analysis of the particles just after heat-shock(80° C. for 15 minutes). The average number of viable bacterial sporesin non-heat-shock (NHS) corncob particles (control) after 25 minutes ofthermal treatment at 150° C. and 155° C. was 4.18 and 4.20 log₁₀ CFU/g,respectively. These results represent decreases of only 1.02 and 1.0log₁₀ CFU/g reductions, respectively, in the initial numbers of viablespores in the corncob particles.

Heat-shock and tempering of corncob particles once (HST1×) or twice(HST2×) prior to drying resulted in drastic reductions in numbers ofviable spores following thermal (drying) treatment at 150° C. and 155°C. (see Table 20). In corncob particles that received one priorheat-shock/tempering (HST1×), average reductions in numbers of viablespores compared to initial number of viable spores were 3.40, 3.65, and3.85 log₁₀ following thermal treatment at 150° C. for 25, 30, and 35minutes, respectively. In comparison, corncob particles that receivedtwo prior heat-shock/tempering (HST2×) had an average reductions ininitial numbers of viable spores of 3.52, 3.73, and 4.09 log₁₀ followingthermal treatment at 150° C. for 25, 30, and 35 minutes, respectively.

Average reductions in initial numbers of viable spores in corncobparticles that received one prior heat-shock/tempering (HST1×) were3.88, 3.89, and 4.24 log₁₀, respectively, after 25, 30, and 35 minutesof thermal treatment at 155° C., compare to reductions of 3.61, 3.77,and 4.05 log₁₀ in initial numbers of viable spores in corncob particlesthat received two prior heat-shock/tempering (HST2×). Differences inLog₁₀ reductions in numbers of viable spores that were not heat-shocked(NHS) or treated with HST1× or HST2× and heated at 150° C. and 155° C.are shown in FIGS. 6A and 6B, respectively.

Non-heat shocked spores exhibited very high heat resistance; log₁₀reductions in NHS spores following heating at 150° C. for 25, 30, 35 and40 minutes were only 1.03, 1.54, 1.96, and 2.45, respectively (6A).Log₁₀ reductions in NHS spores following heating at 155° C. for 25, 30,and 35 minutes were 1.0, 1.33, and 1.62, respectively (FIG. 6B). Therewere very little differences in Log₁₀ reductions of numbers of viablespores treated with HST1× or HST2× and heated at 150° C.; differenceswere 0.12, 0.08, 0.25 and 0.50 Log₁₀ CFU/g, following heating of corncobparticles for 25, 30, 35 and 40 minutes, respectively (FIG. 6A). Similarnegligible differences were observed in Log₁₀ reductions of numbers ofviable spores treated with HST1× or HST2× and heated at 155° C.;differences were 0.27, 0.12, and 0.19 Log₁₀ CFU/g, following heating ofcorncob particles for 25, 30, and 35 minutes, respectively (FIG. 6B).

Differences in Survival in Types of Bacterial Spores Isolated fromCorncob Particles and Exposed to Heat-Shock, Tempering and ThermalTreatment

Corncob particles (⅛″) with 80% or 100% (w/w) added moisture wereexposed to 80° C. for 15 minutes to heat-shock bacterial spores and killbacterial vegetative cells. The heat-shocked particles were thenanalyzed for viable spores by plating samples of diluent (used to removespores from the particles) on dextrose tryptone agar (DTA). Theinoculated DTA plates were incubated at 35° C. for 72 hours beforechecking for different types of bacterial colonies.

Four types of bacterial colonies (called Isolates A, B, C, and D; seeFIG. 7) were selected based on colony morphology (round, irregular,smooth, wrinkled, moist, dry). Each colony was streak plated on DTA.Following incubation of DTA plates, isolated colonies were picked andstreak plated again on fresh DTA plates to ensure isolation of purecultures. The final isolates were suspended in separate tubes of sterile0.1% (w/v) peptone. Aliquots (0.2-ml) of each cell suspension weresurface plated on DTA plates to produce a lawn of cells followingincubation of the inoculated DTA plates at 35° C. or 55° C. for 72hours. The DTA plates were held at 35° C. for a total of 7 days toinduce formation of bacterial spores.

Bacterial spores were collected from the lawn of bacterial growth bypipetting 5.0 ml of sterile saline onto the surface of the lawn on eachDTA plate an gently rubbing that surface with a sterile bent glass rod.The spores were harvested by centrifugation (10,000×g, 10 min, 4° C.) ofthe spore suspensions and discarding the supernatant. The pelletedspores were washed by suspending them in fresh saline by vortexing. Thespore suspensions were subjected to centrifugation and theirsupernatants discarded. The pelleted spores were suspended in sterilesaline. A portion of each spore suspension was subjected to heat-shockand then diluted and plated in DTA to determine the numbers of viablespores from each of the four types of bacterial colonies isolated fromthe corncob particles. This information was used to adjust theconcentration of each spore suspension to obtain ˜10⁷ CFU/ml.

Samples (25-gram) of sterile corncob particles in sterile Erlenmeyerflasks were inoculated with suspensions of bacterial spores to obtain˜10⁵ CFU/g. In each flask the inoculated particles were soaked withfilter-sterilized water, heat-shocked, tempered and heat-treated aspreviously described above (HST1×).

Microbial analysis of corncob particles were analyzed using standardmethods as described above. Inoculated DTA plates were incubated at 35°C. for 72 hours (for mesophilic aerobic spore-formers) and 55° C. for 72hours (for thermophilic aerobic spore-formers).

FIG. 8 shows colonies of bacterial spore-formers on DTA followingincubation at 35° C. for 72 hours. Colonies that were selected anddesignated as Isolates A, B, C, and D are shown in the figure. Isolate Awas a smooth watery colony, whereas, other isolates were generallywrinkled and dry. One of the spore-forming bacteria (Isolate A) from thecorncob particles grew at both incubation temperatures 35° C. and 55° C.The other three isolates (B, C, and D) grew only at 35° C. GenerallyIsolate A exhibited a consistently higher resistance to the treatmentcompared to other isolates; however, there were no substantialdifferences in heat resistance among isolates (Table 21).

TABLE 21 Influence of heat-shock/tempering processes on the heatdestruction of bacterial spores (Isolates A, B, C, and D) in corncobparticles (1/8″) with 80% (w/w) added water. Spore Heat-shock andIsolate tempering 25 min 30 min 35 min 40 min 150° C. final heatingSurvivors (log10 cfu/g) A NHS (control) 4.21 3.48 3.22 2.45 HST1x 1.861.72 1.46 1.04 B NHS (control) 4.06 3.32 2.94 1.60 HST1x 1.64 1.48 1.00<1.0 C NHS (control) 3.98 3.20 2.88 1.79 HST1x 1.66 1.53 1.30 <1.0 D NHS(control) 4.02 3.28 2.85 2.0 HSTlx 1.78 1.60 1.47 1.0 155° C. finalheating Survivors (log10 cfu/g) A NHS (control) 4.26 3.52 3.30 HST1x1.73 1.64 1.29 B NHS (control) 4.00 3.28 2.76 HST1x 1.72 1.50 1.30 C NHS(control) 4.02 3.16 2.54 HST1x 1.54 1.36 1.00 D NHS (control) 4.14 3.082.44 HST1x 1.60 1.48 1.36 Bacterial colonies were counted at 72 hours ofincubation of agar plates.Effect of Water and Selected “Germinant” Solutions on the Extent ofSpore Germination in Corncob Particles

The methods for application of the processing treatment (HST1×) were thesame as previously described except that germinants were added to thewater used for moistening the particles prior to treatment. Corn steepwater was prepared by soaking cornmeal for one hour in water before use.

A summary of the results of three experiments are shown in Table 22below. Only combinations of heat-shock and L-alanine, 0.1% peptone or0.25% corn steep water were highly effective in achieving high levels ofgermination (i.e. as high as 98 and 99%).

TABLE 22 Effect of various germinants on bacterial spore germination incorncob particles. Percent Treatment Germination No heat-shock, nogerminant  5% No heat-shock + L-alanine 76% Heat-shock + water 11%Heat-shock + L-alanine 98% Heat-shock + peptone (0.1%) 99% Heat-shock +corn steep water (0.25%) 98% All heat-shocked particles were tempered at40° C. for 15 minutes.

When L-alanine, 0.1% peptone or 0.25% corn steep water was used in thetreatment process, final heating of the corncob particles at 150° C. or155° C. was most effective in consistently reducing the population ofbacterial spores to less than 10 CFU/g after 30 or 35 minutes of heatingwith only one heat-shock. (See FIG. 9).

Based on the results of these examples, a process for thermaldestruction of microbes in PFR material has been developed. Thesanitizing process subjects microbes, and in particular bacterialspores, in PFR material to conditions where they become sensitive toheating as they germinate and emerge as easily destroyed vegetativecells. The process steps include:

-   -   1. Activation by heat-shock, which prepares spores for        germination. PFR material is completely coated with germinant        enriched water. The germination of bacterial spores is further        activated by increasing the temperature so that each particle is        subjected to 80° C. for 15 minutes.    -   2. Germination and outgrowth of the spores by tempering at        40° C. for about 30-60 minutes. In this step of the process,        spores are germinating or exhibiting outgrowth (producing        vegetative cells) and become sensitive to heat. Generally the        microbial enzymes involved in germination of bacterial spores        work best within a temperature range of 35° C. to 42° C. A        holding time of 40 minutes (minimum) is recommended for        consistency of results because not all spores initiate the        process of germination at the same time.    -   3. Destruction of vegetative cells by drying the PFR material        via subjecting it to 150°—155° C. temperatures until the        moisture level is 20% or below, preferably 10% or below (water        activity level (a_(w)) of approximately 0.368).

Although specific embodiments of the invention have been describedherein in some detail, all such descriptions are solely for the purposesof explaining the various aspects of the invention, and are not intendedto limit the scope of the invention as defined in the claims whichfollow. Those skilled in the art will understand that the embodimentshown and described is exemplary, and various other substitutions,alterations and modifications, including but not limited to those designalternatives specifically discussed herein, may be made in the practiceof the invention without departing from its scope.

What is claimed is:
 1. A method of sterilizing, substantially reducing,or eliminating populations of microbes in a PFR material, comprising:wetting the PFR material; exposing the PFR material to a heat-shockcomprising 65° to 90° C. for 10 to 30 minutes; tempering theheat-shocked PFR material at 35° C. to 45° C.; and drying the PFRmaterial by heating at between about 115° and 177° C. for approximately15 to 40 minutes.
 2. The method of claim 1 wherein said process isperformed in one day or less.
 3. The method of claim 2 wherein saidprocess is performed in 5 hours or less.
 4. The method of claim 1,wherein said PFR material is an animal bedding material.
 5. The methodof claim 4, wherein said bedding material comprises one or more ofpressed wood pellets, wood shavings, recycled cardboard, kenaf, sawdust,wheat straw, barley straw, oat straw, timothy straw, forage straws, orcorncob particles.
 6. The method of claim 4, wherein said beddingmaterial is corncob particles.
 7. The method of claim 1 furthercomprising adding a germinant to the PFR material prior toheat-shocking, wherein said germinant is effective in promotion ofgermination of bacterial endospores.
 8. The method of claim 7 whereinsaid germinant is corn powder in aqueous solution.
 9. The method ofclaim 1 wherein said drying comprises heating said PFR material atbetween about 121° and 155° C.
 10. The method of claim 1 wherein saidheating is performed in a forced-air convection oven/dryer.
 11. Themethod of claim 1, wherein said wetting comprises adding a liquid to thePFR material, wherein said amount is between about 40% and about 300% ofthe weight of said PFR material.
 12. The method of claim 11 wherein saidliquid comprises water that has been enriched with a germinant.
 13. Themethod of claim 12 wherein said liquid is enriched with 0.25% (w/w) cornpowder.
 14. The method of claim 1 wherein said heat-shock is about 80°C. for about 15 minutes.
 15. The method of claim 1, further comprisingholding the PFR material following said heat-shocking, wherein saidholding comprises incubating the PFR material at between about 35° andabout 55° C. for 10 to 30 minutes.
 16. The method of claim 15, whereinthe PFR material maintains an internal temperature of about 40° C. forat least 20 minutes.
 17. The method of claim 1, wherein the PFR materialcontains 1 or less log₁₀ CFU of viable microbes per gram of PFR materialfollowing said heating.
 18. The method of claim 1 wherein said PFRmaterial contains less than 20% moisture content after said drying. 19.The method of claim 18 wherein said PFR material contains less than 10%moisture content after said drying.
 20. A method for sterilizing,substantially reducing, or eliminating populations of microbes in a PFRmaterial, comprising: wetting the PFR material with water and agerminant, wherein PFR material is completely coated with said water andgerminant; exposing said PFR material to a heat-shock comprising 65° to90° C. for 10 to 30 minutes; incubating the PFR material at betweenabout 35° and about 45° C. for 20 to 35 minutes; and heating the PFRmaterial at 176.6° C. for approximately 15 to 20 minutes wherein saidprocess is performed in one day or less.
 21. The method of claim 20wherein said PFR material is an animal bedding material.
 22. The methodof claim of claim 21 wherein said animal bedding material comprisescorncob particles.
 23. The method of claim 20 wherein the PFR materialcontains 10 or less log₁₀ CFU of viable microbes per gram of PFRmaterial mix following said heating.